Mitochondria play a pivotal role in cell survival and tissue development by virtue of their role in energy metabolism, regulation of cellular Ca2+ homeostasis and apoptosis. Given this multifactorial role, these aspects of cellular function must operate as an integrated system. Consequently, mitochondrial Ca2+ homeostasis must be tightly regulated and is based in a series of specific uptake and release systems. Yet, mitochondria can easily undergo an inner mitochondrial membrane (IMM) permeability increase to relatively large solutes called the permeability transition (PT), through the regulated opening of an IMM pore, the mitochondrial permeability transition pore (PTP). A great deal of information is available about the functional properties of the PTP and pathological activation of the PTP can have dramatic consequences on mitochondrial function. As a result, the PTP has long been known to play a key role in mitochondrial dysfunction associated with human pathological events such as ischemia-reperfusion injury and neurodegeneration. However, despite detailed functional characterization over the last 30 years, none of the candidate pore components in traditional models of the PTP has withstood critical genetic tests. In essence then, we have a remarkably poor understanding of the molecular components forming and regulating the PTP. The overall goal of this proposal is to use the pharmacological, biochemical and genetic tools that we have developed for the unbiased identification of proteins involved in the formation of the PTP, followed by a variety of tests to confirm their roles in PTP activity. Since the PTP has been demonstrated to play a critical role in variety of human diseases, we anticipate that the rigorous identification of proteins forming or regulating the formation of the PTP increases our ability to define therapies targeting this important complex of proteins. Our specific plans include: 1) The peripheral benzodiazepine receptor (PBR), which remains the only biochemically identified component included in traditional models of the PTP that has not been subjected to rigorous genetic testing. The goal of this aim is to apply genetic tests of the involvement of the PBR in the formation or regulation of the PTP through conditional elimination of PBR expression. 2) Biochemical data indicate that CyPD binds with high affinity to a limited set of IMM sites and genetic analysis has demonstrated that it is a key regulator of the PTP. Consequently, the goal of this aim is to employ the CyPD molecule in tandem affinity purification strategies for the identification of components of the PTP, followed by rigorous testing of their roles. 3) In higher organisms, a significant fraction of the p66 isoform of ShcA is localized to mitochondria where it binds cyt. c and acts as an oxidoreductase, shuttling electrons from cyt c to molecular oxygen in the creation of reactive oxygen species (ROS). Since ROS are potent inducers of the PTP, the PTP has been proposed to constitute the immediate downstream target of mitochondrial p66 action in the activation of apoptotic pathways. The goal of this aim will be to use of the genetic and molecular tools at our disposal to define the exact relationship between p66-dependent pathways and the PTP, a link that has yet to be critically established. PUBLIC HEALTH RELEVANCE: The mitochondrial permeability transition pore has been studied for over 50 years and has been implicated, for example, in ischemia-reperfusion injury of the heart and brain, muscular dystrophy caused by collagen VI deficiency, and in the axonal damage occurring during MS among many other pathological conditions. Since little is known of the molecular composition of the PTP, our goals in this application are to use the pharmacological, biochemical and genetic tools we have established for the unbiased identification of proteins involved in the formation of the PTP and to use a variety of in vitro and in vivo tests to confirm their roles, either as core components of the pore itself, or regulators of pore activity. Since the PTP is of direct relevance to variety of human pathological conditions, we anticipate that the rigorous and careful identification of proteins forming or regulating the formation of the PTP will increase our ability to define therapies targeting these proteins as treatments for a wide variety of human diseases.